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建立了人血清淀粉样蛋白A(SAA)纯品的液相色谱-同位素稀释串联质谱检测方法。首先采用凝胶电泳和液相色谱法对SAA的纯度进行了表征，然后采用飞行时间质谱对SAA的相对分子量和肽图进行了测定，最后利用氨基酸水解-同位素稀释串联质谱方法对SAA含量进行了准确测定。结果表明，SAA样品纯度为99.39%,实测相对分子质量为11 678.73 Da,与理论分子量11 682.7 Da高度一致，氨基酸序列覆盖率99.04%,氨基酸水解-同位素稀释串联质谱法的定量结果为80.9μg/g,扩展不确定度为2.0μg/g (k=2)。该方法具有较高的精密度和准确度，测量结果可溯源至SI单位，为SAA蛋白标准物质的研制奠定了基础。
Abstract：A liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the detection of pure human serum amyloid A(SAA) was established.The purity of SAA was initially characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography.Subsequently, the relative molecular weight and peptide map of SAA were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The SAA content was then accurately quantified using an amino acid hydrolysis-isotope dilution LC-MS/MS method.The results demonstrated that the purity of the SAA sample was 99.39%,and the measured relative molecular weight was 11 678.73 Da, which was consistent with the theoretical value of 11 682.7 Da.Furthermore, the amino acid sequence coverage rate was found to be 99.04%.The quantitative result of the amino acid hydrolysis-isotope dilution LC-MS/MS method was 80.9 μg/g, with an uncertainty of 2.0 μg/g(k=2).This method exhibits high precision and accuracy, with measurement results traceable to the International System of Units(SI units),laying a solid foundation for the development of SAA protein reference materials.
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基本信息:
DOI：10.13822/j.cnki.hxsj.2024.0498
中图分类号:O657.63;R446.11
引用信息:
[1]朱天翼,李兰英,孟娇然等.人血清淀粉样蛋白A纯品含量的同位素稀释质谱方法研究[J].化学试剂,2025,47(06):21-27.DOI:10.13822/j.cnki.hxsj.2024.0498.
基金信息:
国家市场监督管理总局科技计划项目(2023MK027); 上海市计量测试技术研究院青年科技创新项目(H00RY2403)